GSM6774193: Budding yeast, dSnf2, replicate 2, MNase-seq; Saccharomyces cerevisiae; MNase-Seq
Final 1% formaldehyde was treated for crosslinking at 30oC for 15mins and the reaction was terminated by final 125mM of glycine. Cells were harvested by 3,000rpm centrifugation for 5mins and washed twice by 10mL of 1M Sorbitol. The washed cell pellets were resuspended in 4.785mL of YLE buffer (1M Sortibol and 5mM beta-mercaptoethanol). Check the OD600 of 10uL of cells with 90uL of 1% SDS. Cells were lysed by 225uL of 2mg/mL Zymolase-100T at 30oC with rotating on nutator. Wash the lysed cells twice by 5mL of 1M Sorbitol when the OD600 of 10uL of lysed cells with 90uL 1% SDS was decreased up to 10~20% of initial OD600. The washed cells were resuspended in 1.2mL of SDB buffer (1M Sorbitol, 50mM NaCl, 10mM Tris-HCl pH7.5, 5mM MgCl2, 1mM CaCl2, 1mM beta-mercaptoechanol, 0.5mM Spermidine and 0.075% of NP-40). 1,130U of MNase was treated for 300uL of cells at 37oC and aliquot 100uL of the MNase-treated cells to stop the reaction by adding final 10mM of EDTA and 20mM EGTA. Final 0.12% of SDS and 2.1ug of proteinase K were treated at 65oC for overnight. The DNA was purified by Phenol:Chloroform extraction and 30ug of RNase A at 37oC for 1hr. The sample was purified by Phenol:Chloroform:Isoamyl Alcohol and precipitated by final 0.3M Sodium Acetate and Isopropanol for 5mins at RT. The DNA was collected by 13,500rpm for 30mins at 40oC and washed twice by 500uL of ice cold 80% Ethanol. The DNA were dissolved in 18uL of TE at 37oC with tapping for ~20mins. The purified DNA was ran in 1.5% of 1xTBE gel to take mononucleosomal DNA bands through Qiagen gel extraction kit if the mononucleosomal DNA is 70~80% of the sample. 100ng of mononucleosomal DNA was used for library preparation by Illumina TruSeq DNA PCR-Free kit.